Apparatus and method for isolating and/or analyzing charged molecules

ABSTRACT

Apparatus to isolate and/or analyze charged biomolecules, preferably nucleic acids, comprising: a vessel made substantially of a rigid plastic comprising a receiving chamber to receive reagents, this receiving chamber being externally accessible through an access aperture of the vessel, further two electrodes which can be moved into the receiving chamber to be in contact with the reagents, further comprising, according to the invention: a tube unit made of electrically non-conducting plastic and of such dimensions and/or being designed and/or affixable in such a way that at least part of its inside surface can be made to contact reagents and/or samples contained in the receiving chamber, the tube unit and one of the electrodes being so configured that this electrode can be brought into contact in the tube unit with reagents and/or samples. The apparatus of the invention is especially suitable to carry out methods to isolate and/or analyze charged biomolecules.

The invention relates to apparatus for isolating and/or analyzingelectrically charged biomolecules, illustratively to isolate and/oranalyze nucleic acids, having a vessel made of a substantially rigidplastic and comprising a collection chamber to receive a reactionmixture of sample and reagents, the collection chamber being externallyaccessible through an intake aperture of the vessel, and furthermore twoelectrodes movable into the collection chamber to come into contact withthe reaction mixture.

The invention moreover relates to a method for isolating and/oranalyzing electrically charged biomolecules, preferably nucleic acids,by adsorbing charged biomolecules contained in a solution to anappropriate adsorbent, by separating the remnant solution and optionallywashing the adsorbent, by detaching the biomolecules from the adsorbentand separating the biomolecules, and to using the apparatus of theinvention in the method of the invention.

Apparatus of this species are used in biochemical analysis and in theperipheral field of modern pharmaceutical identification of activesubstances (HST=High Throughput Screening) in the most diverse domains,for instance in nucleic-acid analysis. Illustratively the patentdocument WO 97/34908, of earlier priority than the present applicationbut published after the priority date of the present application,discloses apparatus of the species wherein the vessel besides acollection chamber also comprises a withdrawal chamber communicatingthrough a transfer duct with the collection chamber. Nucleic acids to beisolated are electrophoretically moved from the collection chamberthrough the transfer duct into the removal chamber and there areenriched. Moreover the vessel is fitted at its base wall with a pipestub allowing draining the collection chamber. On the whole the abovedescribed vessel is of complex design entailing substantialmanufacturing cost. Especially as regards applications in which theapparatus cannot or is not allowed to be used again on given grounds,there is a need for a simpler and more economic problem resolution.

The discussion to follow of further documentation relates to furtheraspects of such apparatus calling for improvements and the methods ofthe state of the art used in their operation:

Illustratively further electrophoretic equipment is known using platinumelectrodes. Platinum is highly resistant to redox processes and, beingan inert electrode material, constitutes a durable solution. However theentailed high material costs, for instance of coated electrodes forsingle use, are a drawback. Moreover the coating of platinum surfaces isa complex engineering procedure. Plastic electrodes, for instance madeof synthetic carbon (BIO Fonum Forschung und Entwicklung, GIT Verlag,19^(th) year, vol 12, 1996, p 584) also are state of the art. Howeversuch apparatus are unsuitable for single use because they cannot be madeeconomically, for instance by injection molding. A survey ofelectrophoretic gel methods is described in “Electrophoresis Theory,Techniques; and Biochemical and Clinical Applications”, Ed. A. T.Andrews, ISBN 0-19-854633-5, Clarendon Press, Oxford 1986. Besidesnormal gel electrophoretic methods, also so-called plotting techniquesare described in the above work as well as in a work by Southern (GB 8810400, 1988), which require corresponding plate electrodes. On economicgrounds, platinum electrodes are ruled out entirely. In general V2Asteel may also be used.

The patent documents WO 95/12808 and U.S. Pat. No. 5,605,662 describesystems hybridizing nucleic acids and also employing a special electrodeconfiguration. The heart of these documents is micro-apparatus whereinnucleic acids are hybridized. A special stringency check is carried outby electronic interactions with micro-electrodes. The surfaces of thesemicro-electrodes are made of a material allowing free transport ofcompensating ions. This apparatus is used to concentrate nucleic acidsand to carry out corresponding hybridizations. By appropriately changingthe electrode polarity, poorly hybridizing parts of nucleic acid can beremoved.

The electrode is coated with an oligonucleotide and can be prepared bymicrolithography. The following materials are cited for the electrodes:aluminum, gold, silver, tin, copper, platinum, palladium, carbon,semiconductors and combinations thereof. The manufacture of theseelectrodes or the deposition of the electrodes on certain substancesaccording to the patent documents WO 95/12807 or U.S. Pat. No. 5,605,662as a rule is implemented by vacuum evaporation or evaporation coatingtechniques. If appropriately finished, the electrode surface may becoated with biologically active components such as nucleic acids.

The patent document WO 96/15440 describes another procedure requiringelectrodes. This so-called electrochemiluminescent method generates aluminescence signal by exciting molecules at the surface of anelectrode. Using magnetic fields, illustratively magnetic particlesbearing appropriate luminescence labels are transported to the electrodesurface. The special embodiment in this document is a reusable in-linecell. This cell comprises a preferably gold electrode. The matchingelectrode is a corresponding reference electrode. As a rule the matchingelectrode is a silver/silver-chloride system. All these procedures incurthe drawback of using expensive noble metals for the electrode materialand consequently are unsuitable for instance for a single-use apparatus.

The patent document WO 97/02487 describes a planar device in the form ofa test strip to measure electroluminescence.

A procedure of electro-elution is described in “Methods in Enzymology65” [pp 371-380, 1980]. Platinum electrodes are used.

A method for coating non-conducting plastic surfaces using avidin orstreptavidin is described in the patent documents U.S. Pat. No.4,656,252 and U.S. Pat. No. 4,478,914 Re. 31,712.

The German patent document A1 195 20 398 describes magnetic particleshaving a glass surface and which are suitable for isolating nucleicacids and which may be used in apparatus of the present application. Acomparable procedure employing porous glass surfaces and covalent bondsis described in U.S. Pat. No. 5,601,979 for nucleic-acid hybridization.

The patent document WO 97/01646 discloses a procedure toelectrochemically detect nucleic-acid hybridizations using electrodesconsisting of substrates on a surface. According to the patent documentsWO 92/20702; WO 92/20703; EP A1 0,618,923 and WO 96/27679, furthermoreso-called peptide nucleic acids (PNA) can be advantageously used,besides nucleic acids, in hybridization.

On the other hand it is the objective of the present invention to createapparatus of the initially cited kind which is of simple design and canbe manufactured economically.

The invention solves this problem by apparatus of the initially citedkind which furthermore comprises a tube unit made of an electricallynon-conducting plastic and dimensioned and/or configured and/or mountedin such manner that at least part of its inside surface may be broughtinto contact with the reaction mixture contained in the collectionchamber whereas the tube unit and one of the electrodes are designed insuch manner that this electrode can be brought into contact, inside thetube unit, with the reaction mixture. Because the invention provides thetube unit, the design comprising a vessel with a separately formedremoval chamber can be dropped and as a result the vessel is constructedin commensurately simple manner and manufactured correspondingly moreeconomically. In the invention, the removal chamber is present insidethe tube unit and as a result this tube unit, being a simple component,contributes only insignificantly to the manufacturing costs of theapparatus as a whole.

The tube unit may be configured inside the vessel in widely differentways:

Illustratively this tube unit may be affixed to one of the electrodes,preferably being attached to it, and it may be inserted into the vesseljointly with said electrode. Furthermore, the second electrode may beaffixed to the tube unit, preferably to attach it to this electrode.These embodiment variations are particularly significant regardingapparatus implementing in automated manner the isolation and/or analysisof charged biomolecules while using a robot arm displaced in programmedmanner. By means of this robot arm, the electrode(s) and the tube unitmay be fetched from corresponding magazines, be attached to each otheras desired and be introduced into the vessel.

In addition or alternatively, the tube unit may be affixed to the baseof the vessel. To allow exchange of reagents between the vessel and theinside space of the tube unit, the invention proposes that the rube unitbe fitted with apertures at its lower end, preferably in the vicinity ofthe vessel base and/or adjoining to it. Illustratively the tube unit maybe fitted with feet standing on the vessel base and separated from eachother by slots adjoining the base, the exchange of reagents taking placethrough these slots between the feet.

Furthermore the tube unit may be affixed to, preferably suspended from,the edge of the intake aperture.

As regards automated implementation of the isolation and/or analysis ofthe biomolecules, in particular employing a displaceably programmedrobot arm, the invention proposes fitting the tube unit with positioningarms to stabilize its position within the vessel. As a result, the tubeunit shall reliably always be in the same position relative to thevessel walls and thereby it can be moved accurately into its targetposition with a programmed displacement of the robot arm, for instancein order to insert the electrode into the inside space of the tube unit.

In one of the simplest embodiment variations, the entire inner space ofthe tube unit constitutes the electrode chamber. However and equallywell, an electrode chamber may be partitioned off in the tube unit'sinner space, a communication duct between the electrode chamber and theremnant tube unit inner space being sealed by a semi-permeable membrane.This design prevents the biomolecules displaced to one electrode duringelectrophoresis, and during isolation of nucleic acid for instance tothe anode, from being in contact with this electrode and where theymight undergo redox reactions. In order nevertheless to be able toremove in simple manner the nucleic acids enriched in the inner space ofthe tube unit, the invention proposes fitting the upper end of the tubeunit, besides an intake to the electrode chamber, also with an intake toone of the removal chambers near the electrode chamber. In order not tobe restricted solely to liquid electrophoretic procedures when isolatingand/or analyzing biomolecules, the invention proposes placing a mass ofgel, hereafter merely called “gel”, in the tube unit of which itoccupies the full inside width, whereby gel electrophoretic proceduresalso may be carried out with the apparatus of the invention.

To effectively isolate biomolecules which on account of the proceduralsteps discussed above have accumulated at the vessel base, the inventionproposes flaring the tube unit from the one electrode toward the vesselbase. As a result the “intake region” for electrophoretic isolation ofbiomolecules can be enlarged in the immediate vicinity of the oneelectrode.

An illustrative vessel of especially simple design may be implemented inthat the vessel have a base free of apertures and a sidewall free ofapertures. Such a vessel is manufactured in especially simply and henceeconomical manner and in conjunction with two electrodes makes it easyto carry out electrophoretic processes even in the absence of a tubeunit. Even though the efficiency of electrophoretic isolation ofbiomolecules is less in the vicinity of an electrode than when a tubeunit is used, especially when the biomolecules are to be aspirated fromthe vicinity of the electrode, on the other hand this lesser efficiencymay suffice for some procedures. Accordingly independent protection isclaimed for apparatus of which the vessel's base and one sidewall arefree of apertures.

If the vessel is made of a thermally deformable plastic, for instancebeing made by injection-molding, its manufacture shall be especiallyeconomical.

The vessel base may comprise at least one zone of lesser base thickness.This feature is especially advantageous for detection, for instance whena detector, preferably a photomultiplier, is mounted within the zone oflesser base thickness.

Just as the vessel, the electrodes too may be made of electricallyconducting plastics. On economical grounds it is therefore preferredthat the electrodes, or the at least one electrode, shall bemanufactured from aplastic containing electrically conducting additives.These additives maybe graphite, iron, silver, gold or other metalsand/or their mixtures and/or their alloys.

To achieve good electrical conductivity, the invention proposes that theat least one electrode be of an electrical resistance less than 100 MΩ,preferably less than 1 MΩ. Simple manufacture for instance by injectionmolding is made possible ifthe electrodes, or the at least oneelectrode, are made of thermally deformable plastic.

The electrodes may be configured in different ways in the vessel. Inparticular the electrode mounted in the tube unit may be an elementseparate from the vessel. Furthermore the electrode may be affixeddirectly or indirectly to the vessel and/or to the tube unit, preferablyit shall be clamped in order that its position be determined to theparticular other electrode.

Illustratively, in case enriched biomolecules are removed from thevessel for further analysis, the invention proposes at least oneelectrode designed as a pipet tip or part of such a tip. The whole pipettip may be made of an electrically conducting plastic. However and inequally feasible manner, only part of the pipet tip may be made of anelectrically conducting plastic, for instance when electricallyconducting additives are admixed to a first portion of plastic duringinjection molding and then a second portion of plastic being processedin pure form. Alternatively and furthermore, a separately manufacturedelectrode may be mounted in a pipet tip.

The invention proposes to enlarge the area of reaction of the electrodeby mounting or forming extensions to the outside of the electrode.

Lastly, at least one electrode may be integral with the vessel wall.Illustratively the entire vessel may be made of an electricallyconducting plastic whereby this entire vessel shall constitute theelectrode. However it is equally feasible to admix electricallyconducting additives to spatially limited segments of the plastic duringvessel manufacture, as a result of which for instance the cathode isformed on one side of the vessel and an anode on the other. This designis especially significant for an apparatus without a tube unit and ofwhich the vessel's base and sidewall are free from apertures.

In order to reliably preclude electrical shorts in operation, theinvention proposes a spacer preventing bodily contact between theelectrodes. To achieve as simple as possible a removal of enrichedbiomolecules, this spacer may be tubular, preferably to match the outergeometry of a pipet tip.

In order to implement the insertion of electrodes and spacer into thevessel in the fewest possible steps, the spacer and at least oneelectrode may be made into one unit. Moreover the spacer may be designedto be a pipet tip.

As already intimated several times in the above discussion, theelectrodes may be connected to an external power supply.

In one preferred embodiment of the invention, at least one of theelectrodes and/or the semi-permeable membrane is fitted with a coating.Such a coating makes it possible to bind the biomolecules to be isolatedand/or analyzed to the electrode or to the semi-permeable membrane. Theelectric field enhances the rate of binding and the physical approach ofthe biomolecules to the electrode and/or the semi-permeable membrane.Preferably however the binding shall take place at the semi-permeablemembrane because thereby fewer degradations will be applied to thebiomolecules on account of the redox reactions or the like taking placein the vicinity of the electrode or on account of heat dissipation.

Because of this binding by means of the coating, the biomolecules on onehand are easily isolated by removing the electrode or the membrane fromthe apparatus and on the other hand detection of the bound biomoleculesmay be advantageously carried out in known manner.

If such a biomolecules is a protein, then illustratively a labeledantibody against this protein may be added and the binding of theantibody to the protein can be determined by the label and thereby thepresence of the protein in qualitative and/or quantitative manner.Illustratively detection also may be by means ofelectrochemiluminescence as basically described for instance in thepatent document WO96/15440.

On the other hand the biomolecules also may be a nucleic acid which theexpert again may detect by known methods.

The coating of the electrode and/or the membrane preferably shall bemulti-layer. Preferably the coating shall comprise one or morebiological polymers. Suitable biological polymers in particular aremolecules easily deposited on the surfaces of electrodes or membranesand which furthermore are able to impart direct or indirect, specific ornon-specific binding of biomolecules. Such biological polymers are knownper se. Preferred polymers include proteins, and bovine serum albumin(BSA) again is a preferred protein. BSA in general enables non-specificbinding though upon pertinent pretreatment it may also be able to impartspecific binding.

Such pretreatment may for instance thermal. Such pretreated BSA then canbe used as a binding allowing avidin or streptavidin to form a furtherlayer of biological polymers.

In a further prefeed embodiment of the invention, the biological polymeris a binding-specific protein. Such binding-specific proteins are knownto the expert and they are preferably in the widest sense partners ofspecific binding pairs. The presence of a partner of a specific bindingpair at least in one layer of the coating is another preferredembodiment of the invention. Specific binding pairs also are generallyknown, for instance being pairs of ligand and receptor, this being avery broad definition which also may include the following binding pairssuch as antigen/antibody or antibody fragment, hapten/antibody orantibody fragment, and biotin/(strept)-avidin.

When a partner of a specific binding pair is integrated in the coatingof the electrode or the membrane of the invention, the biomolecules tobe isolated and/or analyzed either is conjugate with the other partnerof the binding pair or else the biomolecules itself is the otherpartner.

An especially preferred multilayer coating is composed of pretreated BSAto which is bound steptavidin. Any molecule conjugate with biotin can bebound to this coating.

On the other hand in a highly preferred manner of the invention,regarding the isolation and/or analysis of proteins, their receptors orantibodies specifically directed at the protein shall be integrated intothe coating.

In a further preferred embodiment, the biological polymer present in thecoating is in the form of at least one nucleic acid or oneoligonucleotide. These molecules too can be bound directly by adsorptionto the electrode or the membrane or else, by means of a specific bindingpair, optionally still in combination with further coating layers suchas BSA.

By means of a nucleic acid or a polynucleotide or oligonucleotidepresent in the coating, it is possible to bind by hybridization nucleicacids such as DNA or RNA to be isolated or analyzed.

Furthermore other coatings able to bind the nucleic acids areappropriate within the scope of the present invention. Preferably suchinclude peptide nucleic acids which were cited already above in relationto the Patent documents WO92/20702; WO92/20703; EP A1 0,618,923 andWO96/27679. The PNAs described therein in principle can also be usedwithin the scope of the present invention.

Aside from the above discussed, preferred embodiments of the invention,or in combination with them, the coating may preferably also containreactive linker molecules in at least one layer. Such linker moleculesallow coupling biological polymers using covalent bonds to the electrodeor membrane. Optionally further layers of preferably biological polymersmay adjoin the biological polymers coupled by linker molecules.

In a further development of the invention, appropriate adsorbents arepresent or can be introduced in the vessel in order to separate thebiomolecules from other substances. Illustratively the adsorbents may besilica gel and/or agarose gel and/or polyacrylamide gel and/orion-exchange substances and/or fiberglass and/or glass particles andmagnetic particles preferably enclosed in glass with special nonwovensadsorbent properties.

High efficiency of isolation of biomolecules by adsorption at anappropriate adsorbent can be achieved provided this adsorbent bedistributed as uniformly as possible in the vessel. When thebiomolecules are removed from the vessel, or when they are analyzedwithin it, on the other hand, they are preferably concentrated at apredetermined site. Accordingly the invention proposes in a furtherdevelopment fitting the vessel with a magnetic field source which can beswitched between an active state wherein it exerts an attractive forceon magnetic particles whhin the vessel and an inactive state wherein itexerts no force, or only an insignificant one, on these particles.Illustratively the magnetic field source may be a permanent magnetand/or a body of magnetizable material. However the source also may bein the form of an electromagnet switched between the active an inactivestates. In both cases, when the magnetic field source is a permanentmagnet and when it is an electromagnet, the switching between the activeand inactive states also may be implemented by the source being movedbetween a near-vessel position corresponding to the active state and aposition remote from the vessel corresponding to the inactive state, forinstance by the source being tipped or shifted away.

If the apparatus of the invention is fitted with a robot armdisplaceable in programmed manner, for instance in the form of an arm ofa pipetting robot then this robot and said magnetic field source may bematched to each other in such manner that the switching between theactive and inactive states is implemented by the pipetting robot alone.Illustratively the robot arm can implement the switching between thesaid active and inactive states by resting against a tipping switch, asetting lever or a tipping lever.

To assure in simple manner the assembly of the apparatus of theinvention in its manifold embodiment variations, the invention proposesthat the pipetting robot besides the pipetting plunger also comprise agripper.

A further concept of the invention relates to a combination structure ofapparatus of the invention whereby the apparatus anodes are connected toone another and the cathodes of the apparatus are also connected to eachother. Furthermore the tube units of the apparatus of the invention canbe combined into one combination structure, whereby all vessels can befitted with an associated tube unit by a single motion of the robot arm.Such a combination structure is especially suitable to carry out seriestests for instance of a plurality of nucleic acid samples. In such acombination structure, the vessels may be combined into onemicrotitration plate.

Moreover the invention relates to a basic unit comprising at least oneseat for an apparatus of the invention and/or a combination structure ofapparatus of the invention. This basic unit may comprise prefabricatedreceptacles for at least one magnetic body or electromagnet. However itmay also be already fitted fully with at least one magnetic fieldsource. Moreover this basic unit may be fitted with at least oneelectric feed conductor to make contact with at least one electrode. Theplurality of applications may be raised further in that the at least oneseat shall be heatable and/or coolable.

Another objective of the present invention is a method of the initiallycited kind. The method of the invention is used to isolate and/oranalyze charged biomolecules by adsorbing charged biomolecules insolution at a suitable adsorbent, by detaching the remaining solutionand optionally washing the adsorbent, separating the biomolecules fromthe adsorbent and separating the biomolecules, said method being carriedout in an apparatus of the invention or in a combination structure ofsuch apparatus of the invention, and at least the detachment of thebiomolecules from the adsorbent being implemented by electroelution.

The scope of the present invention also includes methods wherein theadsorbent already loaded with charged biomolecules is directly insertedinto an apparatus of the invention, as a result of which therefore theprocedural steps of adsorption at an adsorbent and separation of theremaining solution and optionally washing are eliminated. Because thepresent invention foremost applies to the elution and separation of thebiomolecules, the insertion of an adsorbent loaded with biomoleculesalso must be construed as being part of the present invention.

Within the scope of the present invention, “electroelution” means thedetachment of the biomolecules from the adsorbent by an applied electricfield generated between the electrodes in the apparatus. The chargedbiomolecules migrate in the electric field according to the electriccharge and may be accumulated in the vicinity of the oppositelypolarized electrode. Biomolecules may be charged both positively andnegatively. Nucleic acids are charged negatively and will migrate in theelectric field to the anode. Depending on their amino-acid composition,proteins may have a net excess positive or negative charge. Depending onthe biomolecules to be analyzed and/or isolated, the electrodes of theapparatus of the invention may be polarized in such manner that theisolation and/or analysis can be carried out advantageously.

The adsorbent by means of which the charged biomolecules are separatedfrom other substances present in the solution preferably is selectedfrom a gel, an ion-exchanger substance, fiberglass nonwovens and glassparticles, in particular magnetic particles enclosed in glass. The gelsmay be generally known separation gels such as agarose or polyacrylicamide gels as well as silica gels or the like. Preferably, when usingglass particles, the glass surface shall appropriately be pretreated ormodified to bind biomolecules. Such glass particles, mostly called“beads”, are known to the expert and are made by many enterprises.

Furthermore the apparatus of the invention may be used to elute chargedbiomolecules out of a gel by being configured jointly withelectrophoretic buffers between two electrodes of the invention in areaction vessel, the electrophoretic flow resulting in release from thegel.

Following binding the charged biomolecules to the adsorbent, the ionstrength of the solution where necessary may be matched to the adsorbentin the process of electroelution. This matching may be implementedeither by salting the present solution, for instance the last washsolution, or preferably by adding an electroelution buffer ofappropriate ionic strength.

Preferably the biomolecules shall be separated by means of the electricfield in the vicinity of the electrode polarized oppositely to themolecules.

Due to the application of an electric field, the biomolecules migrateout of the adsorbent, i.e. they are detached, and then move toward theoppositely polarized electrode.

Surprisingly and in a preferred embodiment, the detachment issubstantially improved by raising the temperature to 30 to 100° C.,preferably 55 to 80° C.

In another preferred embodiment of the invention, the starting point isnot a solution containing charged biomolecules in their free form, butinstead a solution comprising cells is used, preferably intact cells,containing the biomolecules. These cells are bound to an adsorbent suchas magnetic glass particles. After the cells have been adsorbed, theyare lysed and subsequently released, charged biomolecules will beconcentrated by means of an electric field in the vicinity of theoppositely polarized electrode. Molecules to be isolated from the cellsare, in the invention, especially nucleic acids.

Preferably again, the biomolecules released from the cells shall be oncemore subjected to separation from other, by released molecules beingbound once more to an adsorbent, for the purpose of achieving betterisolation and analysis. The renewed adsorption may be carried out usingthe same but also different adsorbents, depending on the adsorbentspecificity.

Illustratively, in order to isolate biomolecules only from cells of aspecific kind, an adsorbent may be used which comprises receptors forsurface antigens of specific cells. The expert is conversant withfeasible applications and variations regarding the biomolecules to beisolated, and such applications and variations are easily implemented bymeans of the remaining disclosure of the present invention.

Moreover the method of the invention remains unchanged, that is, elutionand separation are carried out as already described above for theprevious configurations.

In a further preferred embodiment of the invention, glass-enclosedmagnetic particles are used as adsorbents and, during elution of thebiomolecules, are kept spatially separate from the biomolecules by meansof the magnetic field source. In particular this feature is implementedin that following biomolecular adsorption, the magnetic field source andthe electrodes simultaneously act in spatially opposite manner on themagnetic particles and the biomolecules in order to carry out elutionand separation. In this process the biomolecules are concentrated in thevicinity of the oppositely polarized electrode whereas the magneticparticles are preferentially collected by the magnetic field source atthe most remote possible distance. In this manner it is possible toavert degradation caused by the magnetic particles when removing oranalyzing the biomolecules.

In one implementation of the invention, namely when isolating thebiomolecules, they will be removed from the vicinity of the oppositelypolarized electrode, and preferably they will be pipetted off. Thesolution of the elution buffer then contains concentrated biomolecules.If apparatus of the invention is used, wherein one of the electrodes orthe semi-permeable membrane is coated to bind the molecules, it is thenpossible to directly remove the electrode and/or the semi-permeablemembrane with the biomolecules bound thereto. In a preferredimplementation of the invention, the electrode per se is a pipet tip andany not yet fully bound biomolecules may then also be pipetted off atthe same time.

When the charged biomolecules to be isolated or analyzed are nucleicacids, the binding by the method of the invention preferably takes placeat a coating containing a complementary nucleic acid of the electrodeand/or semi-permeable membrane, by means of hybridization.

In a further implementation of the method of the invention, the chargedbiomolecules not only are isolated, but they are also analyzed in thevery apparatus of the invention. On one hand this analysis can becarried out by labeling the biomolecules and detecting the label inknown manner, optionally using chemiluminescent methods such as citedabove and detection being by a photomultiplier. Correspondingpublications about similarly applicable procedures already were citedabove.

On the other hand, a problem may arise precisely when analyzing nucleicacids in that often they are present only in minute quantities. Theapparatus of the invention offers the specific advantage that theisolation of nucleic acids and further reactions, in this instancepreferably polymerase chain reactions (PCR), can take place in the verysame apparatus. In an especially preferred implementation of the methodof the invention, following separation of the nucleic acids, thereforeall the reagents needed for PCR are added and the amplification iscarried out in manner known per se. In an especially preferred mode, theapparatus of the invention are used in a basic unit fitted with heatableor refrigeratable receptacles. In this manner the heating and coolingphases taking place during PCR can be controlled in especiallyadvantageous manner. However and in equally feasible manner, individualapparatus may be used and these may be temperature-regulated inappropriate manner.

Following PCR, the amplified nucleic acids preferably are concentratedagain with the help of the electric field and/or are bound to theelectrode and/or semi-permeable membrane. Conceivably as well, again anew tube unit may be inserted after amplification into the apparatus ofthe invention containing a gel by means of which it is possible toresolve the obtained nucleic acid in size by the electric field causingmigration through the gel.

A further application of the invention of the above described apparatusrelates to fully automated electrophoresis which may be used foremost inthe general area of active ingredient identification (High ThroughputScreening). Embodiments of the apparatus of the invention filled withgel-like substance are especially significant. Such apparatus may bedisplaced by the gripper system of an xyz robot. Thereupon, using theelectrode system of the invention, electrophoresis may be carried outand subsequently a corresponding detection to investigate the substancemixture separated according to molecular weight. Such detection forinstance may be carried out by dyeing the bands in a dyeing bath orusing a corresponding optical detection unit such as a fluorometer. Inthis procedure the corresponding modules may be appropriately displacedby the gripper system over the operational surface.

In a variation of the method of the invention, therefore, the procedureconsists in isolating and/or analyzing biomolecules by depositing asample containing them on a separation gel and by electrophoreticallyseparating the biomolecules on the basis of different molecular weights,said procedure being characterized in that it is carried out inapparatus of the invention or a combination structure of such apparaus.The molecules separated by electrophoresis remain in single bands in theseparation gel and can be detected already in the apparatus of theinvention or after removing all or part of the gel from the apparatus.

Another objective of the present invention is to use an apparatus of theinvention, a combination structure of apparatus or an apparatus combinedwith a basic unit to implement the method of the invention.

The invention is elucidated below by illustrative embodiments shown inthe attached drawings.

FIG. 1 is a perspective elevation of the first embodiment of theapparatus of the invention with a separate cathode;

FIG. 1a is a simplified schematic side view (not to scale) of anembodiment of FIG. 1;

FIG. 1b is an elevation similar to FIG. 1a additionally showing thepipetting unit of a pipetting robot;

FIG. 2 is a view similar to FIG. 1 of a modified embodiment comprising acathode in the form of an electrically conducting reaction vessel;

FIG. 3 is a view similar to FIG. 1a of a further modified embodiment;

FIGS. 4-6 are perspectives of modified embodiments;

FIGS. 7-11 are sectional side views of further modified embodiments;

FIG. 12 is a perspective of a further modified embodiment;

FIG. 13 is a sectional side view of a further modified embodiment;

FIG. 13a is a simplified schematic top view of the embodiment of FIG.13;

FIGS. 14, 15 are elevations of electrode embodiment variants;

FIG. 16 is a top view of a combination structure of apparatus of theinvention;

FIG. 16a is a perspective of the combination structure of FIG. 16;

FIGS. 17-19 are sectional side views of further apparatus elucidatingvarious detection modes;

FIG. 20 is a schematic side view of a system to isolate nucleic acids,to amplify and to detect by electrochemiluminescence;

FIGS. 21a, 21 b are plots of the results from Example 4;

FIG. 22 is a block diagram of a control system for full nucleic-acidanalysis using nonwoven fiberglass technology; and

FIG. 23 is a functional diagram to control fall analysis of nucleicacids using magnetic glass particles.

An apparatus of the invention to isolate and/or analyze nucleic acids isdenoted in overall manner in FIG. 1 by 500. This apparatus comprises aplastic reaction vessel 270, a cathode 20 a, an anode 20 b and a spacer280. Both the cathode 20 a and the anode 20 b are made of a plasticcontaining electrically conducting additives such as graphite. Moreoverthe anode 20 b is a tube 290 which as shown in FIG. 1b can be attachedin the form of a pipet tip 290 onto the piston/cylinder unit 300 of apipetting robot 190. A voltage may be applied by means of the electricfeed conductors 260 to the electrodes 20 a and 20 b.

The tubular spacer 280 is made of an electrically non-conductingplastic. The inside diameter of this tubular element 280 is so matchedto the outside diameter of the anode 20 b that these two components canbe telescoped into one another in preferably sealing manner after theyhave been removed form their particular magazines, for instance onaccount of a corresponding, programmed displacement of the pipettingrobot 190. The same relation applies to the outside diameter of thetubular element 280 and the inside diameter of the cathode element 250.Again the outside diameter of the cathode element 250 is selected insuch manner that said element can be inserted from above (at 272) intothe receiving chamber 275 of the reaction vessel 270 without contactbeing made (FIG. 1a).

In the embodiment of FIGS. 1, 1 a and 1 b, the tubular element 280assumes a number of functions. On one hand it acts as a spacerpreventing bodily contact between the electrodes 20 a and 20 b and hencea short. Furthermore its inner space constitutes a trrnsfer duct 150allowing the nucleic acids to migrate into the pipet tip/anode 290 onaccount of their negative charge and the voltage applied across thecathode 20 a and the anode 20 b.

Moreover, in order to assure the tubular elements 250, 280 and 290 areseated in clamped manner, these elements when nesting in each other alsomay be fitted each with snugly fitting cone. Such a design also providesthe required sealing of the assembly at the nesting sites.

Illustratively the procedure to isolate nucleic acids is as follows:First the pipetting robot 190 removes a pipet tip 290 constituting theanode 20 b from a pipet-tip magazine and, in a corresponding furthermagazine, connects it to the non-conductive tube element 280 by means ofa suitable press-fit. Lastly the cathode 20 a is added in a furtherconnecting step. Before assembling the unit of anode-spacer-cathodes,the reaction vessel 270 had been filled by the pipetting robot 190 withsample and reagents. Now the unit of anodes/spacerlcathode together withthe cathode 20 a/250 and optionally also with the spacer 280 is dippedinto said reaction mixture. By driving the cylinder-piston unit 300, thereaction miuuure is sucked out of the reaction vessel as far as into thepipet tip 290, care being taken however that the cylinder-piston unit300 shall not come into contact with the reaction mixture. If now avoltage is applied across the cathode 20 a and the anode 20 b, thenucleic acids contained in the reagents will migrate to the anode 20 b,that is into the pipet tip 290. Upon nucleic-acid enrichment in thepipet tip 290, these reagents contained in the transfer duct 150 of thespacer 280 and of the cathode 250 following controlled expulsion can beisolated and accordingly be prepared for transfer into an analyzer.

To assure that the nucleic acids will accumulate solely in the innerspace of the pipet tip 290, the reaction mixture must be presentexclusively in the inner space of the pipet tip 290 when the voltage isapplied across the cathode 20 a/250 and the anode 20 b/290. Accordinglyat least the pipet tip 290 may not be dipped below the level of thereagents in the vessel 270. Because the reaction mixture was aspiratedas far as the anode 20 b/290 before the electrophoretic procedure began,the spacer 280 need not necessarily dip by its outside surface into thereaction mixture.

Basically the apparatus 500 of the invention also allows canying out gelelectrophoresis. For that purpose an electrically conducting gel 420 maybe present in the inner space of the non-conducting tube element 280,that is in the transfer duct 150, said gel occupying the whole insidewidth of said duct (FIG. 1b). Illustrative, appropriate gels includecommonplace agarose gel or a polyacrylic amide gel for instance inpreparatory and/or analytical gel electrophoresis.

In the invention filiermore, the apparatus 500 also may be used forelectroelution, for instance an adsorbent loaded with nucleic acid beingmounted in the reaction vessel 270. By coating with a correspondingelectrophoresis buffer and filling the full unit of cathode/spacer/anodewith electrophoresis buffer, the nucleic acid can be transferred fromthe adsorbent into the pipet tip 290 after a voltage has been appliedacross the electrical feed conductors 260.

FIG. 2 shows an embodiment variation wherein the reaction vessel 270 ismade of an electrically conductive plastic, whereby it simultaneouslyassumes the function of the cathode 20 a. A sub-assembly of an anode 20b constituting the pipet tip 290 and a tube element 280 made of anon-conducting plastic is inserted into this reaction vessel 270. As wasthe case for the embodiment of FIGS. 1, 1 a and 1 b, care must be paidhere too that following aspiration of the reaction mixture out of thereaction vessel 270 this reaction mixture only has gone as far as infront of the inner space of the pipet tip 290 prior to applying thevoltage across the terminals 260 at the pipet tip 290 in order toprevent nucleic-acid enrichment at the outside of the anode 20 b/290. Inthis instance too a gel with which to cany out gel electrophoresis maybe present in the non-conductive tube element 280.

FIG. 3 shows another embodiment comprising a specially configured,electrically non-conducting tube element 280. This tube element 280comprises an upper, essentially tubular, cylindrical segment 280 aadjacent to the anode 20 b and an adjoining lower segment 280 b flaringconically toward the cathode 20 a. This design makes possible especiallyeffective concentration of nucleic acids that illustratively initiallyare in the vicinity of the base of the reaction vessel 270, on accountof a funnel effect, in a tiny volume 150 a underneath thepipet-tip/anode 290. This space can be evacuated upward by means of thepipet tip 290. Obviously gel electrophoresis can also be carried out inthis design.

As regards the embodiment shown in FIG. 4, the electricallynon-conducting spacer 280 is designed as the pipet tip 290. A speciallyconfigured anode 20 b is inserted at the upper end of this pipet tip 290into its inner space 290 c. Even though in this embodiment the reactionvessel 270 is made of an electrically conducting plastic and thereforesimultaneously acts as the cathode 20 a, it is understood that the pipettip 290 forming the spacer 280 can also be used in a reaction vessel 270housing a separately constituted cathode 20 a. Upon carrying outelectrophoresis, there is enrichment in nucleic acids in the pipet tip290 of the embodiment of FIG. 4.

The embodiment of FIG. 5 differs from that of FIG. 4 only by the designsof the pipet tip and the cathode. As shown in FIG. 5, the pipet tip 290comprises a lower segment 290 a, made of a non-conducting plastic andconstituting the spacer 280 and it further comprises an upper segment290 b integral with the segment 290 a and ofwhich the plastic howeverincludes electrically conducting additives in order that said segment290 b constitute the anode 20 b. Otherwise the embodiment of FIG. 5corresponds to that of FIG. 4.

FIG. 6 illustrates an embodiment variant of the electrical terminal 260of the cathode 20 a when latter is constituted by a reaction vessel ofelectrically conducting plastic. This terminal 260 is formed by areceptacle 440 in a base plate 510 which consists either totally or atleast in the region of the receptacle 440 of an electrically conductingmaterial, for instance a metal. The electrical contact between theterminal 260/440 and the cathode 20 a is assured by bodily contact whenthe reaction vessel 270 is moved into the receptacle 440. Making thebase plate 510 or the receptacles 440 of metal offers the advantage ofbetter contact and of allowing faster thermal regulation of the reactionvessel 270, the latter feature being especially significant whenhandling nucleic acids.

FIG. 7 shows another embodiment of the invention. In this embodiment,the spacer 280 stands by feet 280 c on the base 270 a of the reactionvessel 270. Apertures 280 d are present between the feet 280 c and allowexchange of reagents between the inner space of the spacer 280 and thereaction vessel 270.

A transfer duct 150 runs in the spacer 280 and above this duct the innerspace of the spacer 280 branches out. The linear continuation of thetransfer duct 150 is designed as the anode chamber 410 housing an anode20 b of special configuration. The access from the transfer duct 150 tothe anode chamber 410 is covered by a semi-permeable membrane 360preventing direct contact between the nucleic acids and the anode 20 b.A withdrawal chamber 380 underneath the anode chamber 410 communicateswith the transfer duct 150, and the pipet tip 290 of a pipetting robot190 dips into said withdrawal chamber to withdraw the enriched nucleicacids in front of the semi-permeable membrane 360.

The spacer 280 also may be fitted with positioning arms 280 e to fix theposition of the spacer 280 in the reaction vessel 270, said armsillustratively being laterally braced against the walls 270 b of thereaction vessel 270. However it is just as feasible to suspend thespacer 280 by means of these positioning arms 280 e from the upper edgeof the side wall of the reaction vessel or to clamp them in place therewithout the spacer touching the base 270 a of the reaction vessel 270 byits feet 280 c. In this case the reaction vessel is made of anon-conducting plastic and comprises a separate cathode 20 a.

The spacer 280 in the embodiment of FIG. 7 illustratively may be removedby the pipetting robot 190, using a robot gripper 400, from a magazineand be configured in the reaction vessel. After depositing theelectrically non-conducting spacer 280 in the reaction vessel 270, acorresponding loading of the reaction vessel 270, including the transferduct 150 and the withdrawal chamber 380, as well as a separate loadingof the anode chamber 410, for instance with an electrophoretic buffersolution, may be carried out The gripper 400 need not necessarily be inthe form of tongs able to open and close to seize an object. Equallywell the gripper 400 may comprise a rigid, fork-like seat, by means ofwhich it moves underneath a corresponding collar of the object beforelifting it and thus receiving it.

As already mentioned before, the semi-permeable membrane 360 protectsthe nucleic acids against direct contact with the anode to preventelectrical discharges from the nucleic acids in the vicinity of theelectrode.

Essentially the embodiment of FIG. 8 corresponds to that of FIG. 7 fromwhich it merely differs in that a gel 420 with which to carry out gelelectrophoresis is present in the transfer duct 150. This gel projectsdownward from the transfer duct 150 and in this manner dips with a largesurface into the reaction mixture in the reaction vessel 270. The spaceabove the gel 420 can be loaded with reaction mixture through thewithdrawal chamber 380. To reliably preclude throughout the entire innerspace of the spacer 280 that gas bubbles should accumulate anywhere,advantageously, and as shown in FIG. 9, the semi-permeable membrane 360shall not be mounted horizontally but shall assume a given sloperelative to the horizontal to allow the gas bubble to escape through theremoval chamber 380.

The apparatus 500 shown in FIG. 9 comprises another feature in that apipet tip 290 made of an electrically conducting plastic is used as theanode 20 b. This anode 20 b is placed first in the anode chamber 410 tocarry out the electrophoresis and following enrichment and turning OFFthe voltage is the moved by the pipetting robot 190 into the removalchamber 380 to withdraw the nucleic acids which have accumulated betweenthe membrane 360 and the gel 420.

Moreover the reaction vessel 270 shown in FIG. 9 is made of electricallyconducting plastic and mounted in a metallic seat 440.

FIG. 10 shows an especially simple embodiment of an apparatus 500 of theinvention wherein a non-conducting spacer 280 is not used. A cathode 20a and an anode 20 b each with integral electrical terminals 260 aresuspended in a reaction vessel 270 fitted with a base 270 a free ofapertures and side walls 270 b also free of apertures. The reactionvessel 270 is made of electrically non-conducting plastic, whereas theelectrodes 20 a and 20 b besides the electrical terminals 260 are madeof plastic containing electrically conducting additives. The apparatus500 of FIG. 10 allows especially economical manufacture and isespecially suitable to carry out electrophoretic procedures for whichmodes a lower process efficiency is acceptable.

FIG. 11 shows amodified embodiment of the apparatus 500 of FIG. 10wherein the reaction vessel 270 is designed in the manner of a reactortube. Bodily contact between the electrodes 20 a and 20 b is preventedby a spacer 30. Preferably the spacer 30 shall be a hollow cylinder toallow loading the reaction tube 270 through the spacer 30 with reactionmixture or so that the reaction products may be evacuated from thereaction tube by means of a pipet tip passing through the spacer.

A further modified embodiment is shown in FIG. 12. In this apparatus 500the spacer 30 and the two electrodes 20 a and 20 b as well as theelectrode terminals 260 are one unit. Furthermore the hollow-cylindricalspacer 30 is designed in such a way that it is possible to move a pipettip 290 through it to evacuate substances from the reaction vessel 270.In a further variation of this embodiment, the pipet tip 290 may be apart of the unit of electrodes and spacer in that it shall be integralwith the spacer or shall replace it

In the embodiment of FIG. 13 the electrodes 20 a and 20 b are integrallymolded with the reaction vessel 270 made of non-conducting plastic bymeans of a corresponding localized addition of electrically conductingadditives. Magnetic particles 65—more precisely, magnetic particles 65coated with a special glass to adsorb nucleic acids or the like—areconfigured in the reaction vessel 270. Following adsorption of thenucleic acids or the like at the magnetic particles 65 and optionallyany cleaning steps, the substances adsorbed on the magnetic particles 65can be detached from them by pulling them electrophoretically toward theanode 20 b whereas the magnetic particles 65 are attracted by a magnet60 toward the cathode 20 a. Following substance detachment, thesesubstances may be investigated for instance using a photomultiplier 50or another appropriate detector.

The magnet 60 in FIG. 13 is composed of a body of permanent magneticmaterial or of magnetizable material. Electromagnets may be used just aswell as shown in simplified, cross-sectional, diagrammatical manner by60′ in FIG. 13a. The permanent magnet 60 in FIG. 13 is shown in itsactive state, that is a state wherein it is near the reaction vessel 270and thereby can exert adequate attraction on the magnetic particles 65.To be switched into the inactive state, the magnet 60 only need beingmoved away from the reaction vessel 270, for instance by relocation,shifting or tipping away. This motion illustratively may be implementedby the pipetting robot 190. In case the electromagnet 60′ is used,switching into the inactive state can be implemented by turning OFF thepower to the electromagnet 60′.

FIG. 14 shows an anode 20 b designed as a pipet tip 290. This anode 20 bis fitted at its outside with attachments to 450 enlarge the contactarea with the reaction mixture and hence to improve the electroncurrent. At the same these attachments 450 may also be used foragitation or mixing purposes.

FIG. 15 shows an electrode 20 coated with biological polymers. Thecoating comprises several layers 34, 35 and 36 to implement appropriateadhesion to the surface of the electrode 20 a or 20 b. Illustrativelythe layer 34 may comprise biotinylated bovine serum albumin, the layer35 may comprise streptavidin or polystreptavidin, and the layer 36 maycomprise a biotinylated oligonucleotide. The binding of theoligonucleotide to the electrode 20 a or 20 b so generated can beutilized in a way that, with the help of the electric field generatedbetween the electrodes 20 a and 20 b, the hybridization of nucleic acidsshall be facilitated or to improve corresponding electrostringency.

The semi-permeable membrane 360 also may be fitted with a correspondingcoating. This feature offers the advantage that corrosive chemicalsgenerated by the electrophoretic redox process at the electrode surfacecannot interact with the coating because this membrane is configuredsufficiently far away. However the electrophoretic current remainsdirected at the membrane and thereby the desired concentration procedurewill take place.

FIGS. 16 and 16a resp. show simplified diagrammatical top andperspective views of a combination structure 550 of apparatus 500 of theinvention. The cathode and anodes of these apparatus 500 also shown insimplified schematic manner are connected in parallel by buses 80 andare jointly connected to electrical feed conductors 260. The combinationstructure 550 comprises a base plate 510 into which are fitted aplurality of receptacles 440. The buses 80 furthermore are imbedded inthe base plate 510.

Be it borne in mind that the embodiment of the base plate 510 shown inFIGS. 16 and 16a is especially suitable to receive reaction vessels withintegrated cathodes 20 a and anodes 20 b of the embodiment of FIG. 13. Acorresponding combination structure 550 of apparatus 500 however alsomay be used for the case when the reaction vessels 270 consist of anelectrically conducting plastic and thereby form one of the electrodes,preferably the cathode 20 a In that case the entire base plate 510 maybe made of metal as already discussed above in relation to FIG. 6. Ifthe reaction vessels 270 are made of non-conducting plastic, then ametallic base plate 510 is desirable to implement rapid-responsetemperature regulation of the reaction mixtures contained in thereaction vessels 270.

The tube units 280 also may be assembled in a corresponding combinationstructure, preferably into the “96-well” microtitration plate orsub-units derived therefrom, for instance rows each of 8 or of 2×8 tubeunits. This configuration offers the advantage, in addition toeconomical manufacture, of simpler and more rapid handling ordisplacement by means of the robot arm, resulting in a higher sampleprocessing rate. These advantages also apply to a combination structureof pipets or pipet tips.

As shown in FIG. 17, the investigation into the substances contained inthe reaction vessel 270 can also be carried out, using an appropriatedetector 50, for instance a photomultiplier, from the base 270 a of thereaction vessel 270. Therefore in FIG. 17 the reaction vessel 270comprises a zone 270 a 1 of lesser base thickness. Detection can becarried out in especially problem-free manner in this zone, inparticular when using optical detectors.

FIGS. 18 and 19 show embodiments of reaction vessels not part of thepresent invention. However the snapin lids 90 provided therein, forinstance a snap-in lid with an optical window 100 or a snap-in lid 90with a septum 110 pierced by a needle 120, also are applicable in theapparatus 500 of the invention, namely in the reaction vessels 270 ofthe invention.

FIG. 20 shows a complete system to carry out nucleic-acid analysis withelectro-elution, electro-amplification and electrochemiluminescencedetection. This system comprises a pipetting robot 190, a power supply180, one or more displaceable pennanent magnets 60 a and 60 b, aphotomultiplier 50, a receptacle 200 for the apparatus 500 of theinvention, and a rapid-response temperature-regulating system to heat orcool the receptacle 200, such as are known to the expert as temperaturecyclers. All these components are computer controlled and allow completeand fully automated nucleic-acid analysis including nucleic-acidisolation, amplification and detection.

Even though FIG. 20 shows an apparatus of FIG. 19 which is not of theinvention, it is understood that instead of said apparatus in FIG. 19,the above described apparatus 500 of the invention can immediately beused in the system.

FIGS. 21a and 21 b show the test results using electrically conductingpipet tips as the electrodes. The trigger voltage, in this instance 50v, is shown in the lower part, and the output signal of a luminometer(LKB 1250) at an output voltage of about 1 v is shown in the upper part.FIG. 21b shows the light emission dependency on the trigger voltage whenusing electrically conducting pipet tips as electrodes.

Polyethylene, polypropylene, polycarbonates, polystyrene or the like maybe used as thermally deformable plastics.

Generally implementations using from 1 μl to 100 μl are appropriate.Implementations based on reaction volumes up to 100 ml and yieldingfinal volumes of 5 μl to 50 μl and therefore of high concentrationeffectiveness are especially preferred.

The various above discussed embodiments are considered again below withspecial attention paid to aspects of process-technology:

FIG. 11 shows a is simple apparatus to carry outelectrochemiluminescence measurements (Example 3).

FIG. 11 shows a simplified apparatus with a reaction tube 270 and twoelectrodes 20 a, 20 b made by injection-molding. Preferably the reactiontubes are made of such deforming plastic as polyethylene, polypropylene,polycarbonate, polystyrene orthe like and accordingly are electricallynon-conducting. The molded body 30 and the reaction tube are made of thesame material and said body also is insulating. It acts as a spacer forthe electrodes 20 a, 20 b and, in case the reaction vessel contains noconducting liquid, no electrically conductivity arises between theelectrodes. The molded body 30 is cylindrical and hollow, and as aresult the reaction tube can be loaded with the reaction solution, forinstance for electrochemiluminescence. The electrodes also are made ofthis material, however they additionally contain electrically conductingadditives imparting conductivity to segments of the apparatus. Graphiteis preferred as an appropriate additive, however other metallic andelectrically conducting particles or substances such as iron, silver,gold, platinum as well as their mixtures or alloys also are suitable.The resistances of the electrodes or conducting segments typically areless than 100 megohms, preferable less than 1 megohm. The resistance maydrop when a voltage is applied, without the procedure being therebydegraded. By applying a voltage across the electrodes, biologicalprocesses such as electrochemiluminescence for instance may beinitiated. Using appropriate test equipment, light emission may bedetected as being a signal. Other biochemical processes may be carriedout in such apparatus, the above described economical manufactureforemost being suggested for those analytical processes precludingcontamination on account of repeat use of the apparatus.

FIG. 15 shows the configuration of a coating of the electrodes of theinvention (Example 2).

Hybridizations of nucleic acids are another preferred application. Inthis regard electrodes coated with biological polymers areadvantageously used. Preferably the coating may consist of severallayers 34, 35, 36 as shown by FIG. 15 to permit correspondingly strongadhesion to the surface. Illustratively the layer 34 may be abiotinylated bovine serum albumin, layer 35 may be a streptavidin orpolystreptavidin and layer 36 may be a biotinylated oligonucleotide.

The binding so produced of the oligonucleotide to the electrode 20 canbe exploited to facilitate hybridization by means of the electric fieldgenerated by 20 a and 20 b or to improve the correspondingelectrostringency. Such procedures are known to the expert andillustratively are described in the patent documents U.S. Pat. No.4,478,914; EP 0,331,127; EP 0,344,578 or U.S. Pat. No. 5,605,662.

FIG. 13 shows an embodiment mode for a simple injection-molded apparatusfor electrochemiluminescence measurements.

FIG. 13 shows apparatus of the invention of a reaction vessel 270,preferably in the form of an injection-molded component with integratedelectrodes 20 a, 20 b, a reaction space 275 and an aperture 272 abovewhich is located also for instance a photomultiplier 50 detectingluminescence. Conductive plastics in the form of electrodes 20 a, 20 bare integrated into the opposite sides though in electrically insulatedmanner. Illustratively this embodiment also includes a permanent magnet60 which is moved near in such manner to the electrodes that magneticparticles which for instance bear the ruthenium molecules that are to bedetected shall be magnetically attracted to the electrode surface andare excited by an electric trigger into luminescence and shall bedetected by the photomultiplier. This integrated design allowseconomically making this injection-molded part into a disposable itemfor single use.

FIG. 13a sectionally shows the apparatus of the invention with theelectrodes integrated into the molded body and electrically insulated bynon-conducting plastics.

Such a device is appropriate for a large number of applications.Illustratively magnetic particles described in the German patent 44 20732, Example 1a, 1b, which are coated with cell surface antigens toseparate a special cell population, may be used in this embodiment modeof the invention. For this purpose the feed of reagents as well as therequired washing procedure is made possible through the aperture 272.Thereupon lysing reagents are introduced, preferably in the mannerdescribed in the European patent document 0,389,063, which releasenucleic acids from cells. By means of electrophoretic forces, which areexerted over the integrated electrodes, the nucleic acids are separatedfrom the magnetic particles. As shown in FIGS. 13 and 13a, thisprocedure requires configuring the anode 20 b opposite the permanentmagnet 60, so that the negatively charged nucleic acids shallconcentrate in the space in front of the anode while the magneticparticles shall separate at the opposite side.

A comparable application follows from using magnetic particles in themanner disclosed in the German patent 195 20 398. The isolationdescribed therein of nucleic acids is advantageously carried out inapparatus of the invention shown in FIGS. 13 and 13a. Following lysingwith chaotropic salts and binding these special magnetic particles tothe glass surface, the invention allows implementing detaching thenucleic acids from the surface and hence their elution by using thepermanent magnet while at the same time a voltage is applied across theelectrodes.

As a result there is enrichment of magnetic particles at the sideopposite the anode while the nucleic acids are kept in the space infront of the anode. This process is called “electroelution” based on“Methods in Enzymology 65” pp 371-380 [1980].

FIGS. 16 and 16a show an embodiment of apparatus for theelectrochemiluminescence measurement in the form of a simpleinjection-molding part of the 96-well format of the microtitration plate(top view and perspective view).

FIGS. 16 and 16a show one embodiment mode of this apparatus in the formof a micro-titration plate, wherein the electrodes of the individualbowls are connected in electrically conducting manner with correspondingplastics, as a result of which an electrical terminal to connect to thepower supply is present for the whole microtitration plate at two sitesand the electrical feed conductors 80 are integrated into theinjection-molded part. FIG. 16a shows this configuration from above.This apparatus offers the advantage that the previously describedapplications can be carried out in this format of microtitration plate.

FIG. 18 shows an embodiment mode of an apparatus combiningelectroelution and measurement of electrochemiluminescence in the formof a simple injection molded part, in particular as a “closed system”(FIG. 19) reducing contaminations.

FIGS. 18 and 19 show apparatus combining electroelution in a derivativevariation and the measurement ofelectrochemiluminescence. This featureof the invention offers the advantage that the two important processes,namely “nucleic-acid isolation” and “nucleic-acid detection” can becarried out in one and the same apparatus. In other words, by minimizingthe transfer steps of the reaction liquids, substantial reduction incontamination, that is introduction of undesired nucleic acid from thesurroundings, which may lead to spurious analytical results, is madepossible.

The molded body 270 of FIG. 18 corresponds to the design of FIG. 13.However said body comprises several apertures 272, 130, 140 partlysealed by snap-in lids 90, screw caps orthe like. A snap-in lid 90 withan optical window 100 is used to seal the aperture 272. On the otherhand the aperture 130 is fitted with a snap-in lid 90 comprising aseptum 110 which may be pierced by a corresponding needle 120. Theaperture 272 preferably is used to load the reaction space 275 withreagents, the aperture 130 to load the reaction space 160 and theaperture 140 to evacuate by aspiration the two reaction spaces 275, 160.

Preferably magnetic particles to isolate nucleic acids are used in theapparatus of FIG. 18. These magnetic particles may be moved by means ofthe permanent magnet 60 toward the electrode 20 a. Then electroelutioncan be carried outto remove the nucleic acid from these magneticparticles. The nucleic acid migrates through the duct 150 toward theelectrode 20 b. A corresponding supply of reagents can then take placethrough the needle 120, for instance for purposes of amplification,whereas solutions likely to be discarded can be aspirated through theduct 140. The entire apparatus must be cyclically heated and cooled toattain amplification, for instance by the polymerase chain reactionprocess (U.S. Pat. No. 4,683,195).

This procedure preferably is carried out at the apparatus' long sides toattain high heat exchange. Then a second kind of magnetic particles canbe added through the needle 120 to bind the nucleic acids to beamplified. Furthermore corresponding buffers may e used to allowthereupon detecting the amplified nucleic acids for instance byelectrochemiluminescence. For that purpose the magnet 60 is moved nearthe corresponding reaction space 160 and as a result these magneticparticles are attracted toward the electrode 20 a. Then a correspondingelectric trigger can be applied to the integrated electrodes to initiateelectrochemiluminescence.

In the invention, an apparatus of FIG. 18 can be manufactured in abinary injection molding procedure in simple and economical manner andas a rule shall be used only once.

FIG. 19 shows a comparable variation. In this design the photomultiplieris situated underneath an optical window 100 covering the region of thereaction space 160 around the electrode 20 b. A first displaceablepermanent magnet 60 b is situated at this electrode. Contrary to thecase of FIG. 18, a second permanent magnet 60 a for the reaction space275 is situated at the electrode 20 a. The aperture 140 leading to avacuum pump is used to evacuate used reagents and to supply washsolutions. As already described before, the aperture 130 is fitted witha snap-in lid 90 or the like, and so is the aperture 272. This apparatusallows isolating nucleic acids in the manner previously described. Ifappropriate magnetic particles are used, the nucleic acid will bind tothem; by opening the snap-in lid 90 at the aperture 80, anelectroelution buffer can be added. Following application of thepermanent magnet, the magnetic particles are attracted to the electrodes20 a and by applying a corresponding voltage to the electrodes 20 thenucleic acid can be detached from a corresponding particle surface andbe transferred into the reaction space 160. Instead of magneticparticles, other solid phases may be used, such as membranes preferablymade of nylon, nitrocellulose or the like, various papers or non-wovens,foremost with fiberglass contents, non-wovens made 100% of fiberglass ormaterials with ion-exchange active surfaces. In that case amplificationcan be carried out by appropriate steps in the reaction chamber 160around the electrode 20 b. The generated amplicons then can be bound byadding an appropriate second kind of magnetic particles through theaperture 130 while the snap-in lid 90 is open. The magnetic particlesare attracted to the electrode by resting the permanent magnet 60against the reaction space 160. When the snap-in lid 90 is open, anexchange of buffers can always take place through the aperture 140.Finally a special electrochemiluminescence buffer must be added tosubsequently carry out electrochemiluminescence by applying a voltage tothe electrodes 20.

The correspondingly emitted light is detected by the photomultiplier 50as shown in FIG. 18. This embodiment offers the advantage that the twopermanent magnets assure a simple reaction procedure. Furthermore othersolid phases may be used, in which case the second permanent magnet atthe reactions pace 275 may be eliminated.

FIG. 20 shows a complete system to carry out analysis of nucleic acidsusing electroelution, amplification and electrochemiluminescencedetection. This system is composed of an xyz arm of a pipetting robot190 (for instance made by TECAN), a power supply 180, a pump 170 todispose of waste solutions, one or more displaceable permanent magnets60 or 60 a and 60 b, a photomultiplier 50 a receptacle 200 receiving theapparatus of the invention such as are illustratively described in toFIGS. 18 and 19, and rapid heating or cooling means for the receptacle200 such as are foremost known to the expert as temperature cyclers (EPA 0,488,769). All apparatus modules are operated by a correspondingcomputerized control and permit complete, fully automated nucleic-acidanalysis consisting of isolation, amplification and detection of nucleicacids. A septum-piercing needle is shown in FIG. 18, however proceduresto automatically open and closing vessels such as are described in theGerman patent 44 122 86 and apparatus similar to that shown in FIG. 19,may also be used. Typical procedures are elucidated in Examples 4a and4b and in the functional diagrams of FIGS. 22 and 23. FIG. 22 shows afunctional diagram to control complete nucleic-acid analysis usingnonwoven fiberglass technology, whereas FIG. 23 shows a functionaldiagram to control complete nucleic-acid analysis using magnetic glassparticles.

FIGS. 21a and 21 b show test results using electrically conducting pipettips acting as the electrodes 20 from Example 4. In the lower part thetrigger voltage in this case is 50 v, the upper part shows the output ofa luminometer (LKB 1250) at an output of about 1 v. FIG. 21b shows thedependence of light emission on the trigger voltage when usingelectrically conducting pipet tips as the electrodes.

The embodiments discussed below share the feature that they can benested in each other by Cartesian displacement in the xyz direction.

FIG. 1 is a perspective of an embodiment mode of the invention withnesting electrodes in the form of a pipet.

FIG. 1 is an overview summarizing a functional assembly. This assemblyconsists of a reaction vessel 270, an electrically conducting,cylindrical electrode 250 fitted with pertinent electrical feedconductors 260. In the invention these conductors are made of plasticcontaining electrically conducting additives. The diameter of theelement 250 is selected in such manner that it can be inserted in acontactless manner into a reaction vessel 270. The element 280 is anon-conducting connector or a transfer duct. This element 280 can beinserted by clamping into the element 260. A further element 290 isclamped into the upper end of the element 280 and consists of plasticscontaining electrically conducting additives and a correspondingelectrical terminal 260. The cylindrical elements also may be fittedwith a press fit cone to allow corresponding clamping. It was found insurprising manner that using a conventional xyz pipetting robot, it ispossible to assemble an integrated electrode system by first seating theelement 290, then the element 280 being made to dock by an appropriateclamping action and thereupon the element 250 being nested in element280. In this manner one may implement an apparatus with an xyz robot tocarry out electrophoretic processes. Following dipping this nestedapparatus composed of 290, 280 and 250 into the reaction vessel 270,electrophoresis for instance can be carried out wherein the liquidmaterial is moved from 270 through the cylinder 280 toward the electrode290. The electrically non-conducting cylinder part 280 again may containa liquid, though also an electrically conducting gel, so that gelelectrophoresis is made possible in this way too. In the invention, thisapparatus may be used for electroelution, an adsorbent loaded withnucleic acid being situated in the reaction vessel 270 for instance. Bycoating with a corresponding electrophoretic buffer and filling theentire apparatus 250, 280 and 290 with electrophoretic buffer, and uponapplying a voltage to the electrical conductors 260, the nucleic acidmay be transferred from the adsorbent through the three elements in theupper region 280.

Variants of this embodiment are derived below:

FIG. 2 is a perspective of an embodiment with an electrically conductivereaction vessel assuming the function of the cathode.

A reaction vessel 270 is used in FIG. 2 that comprises electricalconductors 260 and is made per se of a plastic with electricallyconducting additives. These then represent a cathode. A correspondingelectrically non-conducting cylinder part 280 can be for instance beattached by an xyz robot to a cathodic electrode 290 and be dipped intothe reaction vessel 270. In this manner electrophoresis of liquids inthe reaction vessel 270 can be implemented in that, after acorresponding voltage has been applied across the anodes and cathodes,transfer of molecules takes place through the transfer duct 280 and acorrespondingly purified substance is removed at the upper end of 280.

FIG. 1a elucidates a cross-section of such apparatus. The reactionvessel 270 is situated at the lower end and may receive an electricallyconducting cylinder part 250 which then illustratively acts as acathode. A corresponding non-conducting cylinder part 280 is thenattached to the cylinder part 250 which in turn is connected directly byan electrically conducting cylinder part 290. In this manner anelectrophoretic current can be set up between the cathode 250 and theanode 290 through the transfer duct 150. The transfer duct may be filledwith the pertinent liquids or also with appropriate gel. The expression“gel” herein denotes a conventional agarose gel or pertinent polyacrylicamide gels for instance for a preparatory gel electrophoresis.

FIG. 1b shows apparatus comprising an appropriate pipet. The pipet isable to load the cavity of the cylinder part 250 and the transfer duct150 in case a liquid is kept there. The pipet furthermore is fitted withcorresponding receptacles for power terminals able to transfer anelectrophoretic voltage to electrical feed conductors 260.

FIG. 3 is a cross-section of a detail of an embodiment acting as aconcentrator.

FIG. 3 is a special embodiment of the electrically non-conductingcylinder part 280. It is fitted with an inverted-funnel shape. Anappropriate concentration may be achieved in this manner. Nucleic acidsfor instance at the base of the reaction vessel 270 may be collectedunderneath the anode 290 in a very small volume. This space may beemptied upward by means of the above described pipet and thecorresponding aperture.

FIG. 10 shows a section of a further embodiment of electodes that areclamped to a reaction vessel. This reaction vessel 270 is electricallynon-conducting and is entered by two electrodes 20 a, 20 b withcorresponding terminals for the electrical feed conductor 260. The twoelectrodes are made of plastic with electrically conducting additivesand can be clamped to the reaction vessel. In this manner a reactionvessel can implement electrophoretic processes any time at the inside.

FIG. 12 shows a further embodiment comprising an electricallynon-conducting reaction vessel 270 and an apparatus consisting of twoelectrically conducting electrodes 20 a, 20 b with correspondingterminals 260 and a non-conducting cylindrical element 30 supporting thetwo electrodes. This cylindrical element 30 is designed in such mannerthat for instance a pipet tip 290 can be attached to this element. Forthat purpose the cylindrical shape may be replaced by a correspondingfrusto-conical one. Thereupon this apparatus may be moved for instanceby an xyz robot into the reaction vessel 270. After a voltage has beenapplied across the two electrodes, local separation of the reactionmixture may be carried out and, using the pipet tip 290, biologicalsubstance can be removed in the immediate vicinity of the correspondingelectrode.

FIG. 5 shows apparatus with a conducting reaction vessel and a pipet tipwith integrated electrode.

FIG. 5 shows a comparable embodiment. Therein the reaction vessel 270 ismade of an electrically conducting plastic, as a result of which thewalls may serve as the cathode. An electric feed conductor 260 isprovided for that purpose. A special pipet tip 290 dips into saidreaction vessel and is fitted at its top part with an integrated plasticcontaining conducting additives, this tip illustratively serving asanode 20 b. In this case the corresponding receptacle for the pipet canbe made metallic and act as the electric feed conductor 260. Anelectrophoretic process may be initiated in this apparatus, followingfilling the pipet tip with electrophoretic buffer and dipping the tipinto the solution of the reaction vessel 270, by applying a voltageacross the cathode and the anode 20 b by means of the feed conductor ofthe reaction vessel 270 and the feed conductor 260 of the pipet tipreceptacle. Following separation of the biological substance, the pipettip is removed from the reaction vessel and shelters the desiredbiological substance in its inner part.

FIG. 6 shows apparatus with a conducting reaction vessel and receptacleand further a pipet tip with integrated electrode.

FIG. 6 illustrates a further detail of the embodiment of the inventionwherein the reaction vessel per se consists of an electricallyconducting plastic and acts as an electrode. The power is appliedthrough an electrically conducting, preferably metallic receptacle 440.The metallic form offers the advantage of the invention that thereaction vessel can respond rapidly to temperature regulation in amanner important in handling nucleic acids.

FIG. 7 shows apparatus with an element 280, a conducting reaction vesseland a pipet tip with affixed electrode.

As shown in FIG. 7, the cathode 20 a enters a reaction vessel made ofnon-conducting plastic 270 and similarly to the embodiment of FIG. 10can be attached to the wall. An electrically non-conducting cylindricalpart 280 with a pertinent Y branch enters said reaction vessel and restson the base by means of suitable foot-like elements, for instance in theform of a tripod. The linear continuation of the transfer duct 150 iscovered by a semi-permeable membrane 360. A space 410 is present abovethe semi-permeable membrane and is entered by an electrode 20 b forinstance acting as the anode. In the invention this electrode is made ofa plastic containing electrically conducting additives. A pipet tip 290enters an evacuation space 380 from which the corresponding materialscan be removed from the inside volume of the reaction vessel 270 throughthe transfer duct 150.

The element 280 can be removed, by an xyz arm of a suitable apparatus400, from a supply magazine and be moved into the reaction vessel. Theapparatus element 400 consists of a fork-like element which may receivethe Y element in appropriate manner. After the electricallynon-conducting cylinder part 280 has been deposited in the reactionvessel, loading can be carried out both through the feed 380 and thefeed 410 which represents a corresponding reaction space 410 above asemi-permeable membrane 360. The semi-permeable membrane 360 is aprotective device mounted in front of the electrode and as a resultelectrical discharges from molecules to be analyzed may be avoided inthe vicinity of the electrode. Illustratively the electrode 20 b made ofelectrically conducting plastic is attached by means of a correspondingreceptacle against an electrically conducting pipet tip, as a result ofwhich the receptacle of the pipet tip 290 acts as the electrical feedline for the electrode.

FIG. 8 shows apparatus with the elements of FIG. 7, but with preparatorygel.

FIG. 8 shows that the transfer duct 150 can be filled with gel and thatin this way gel electrophoresis can be carried out. It is determinant inthis respect that the gel project form the transfer duct in order toachieve optimal dipping into the liquid to be analyzed in the reactionvessel. This protrusion is represented by the element 420 in FIG. 8. Thefeet of the electrically non-conducting cylinder part 280 are not to beconstrued having a moving function but to be a tripod whereby the airmay undergo a corresponding displacement. The air space above the gel420 can be filled through the withdrawal space 380, the configurationbeing such that no air bubbles shall be generated underneath thesemi-permeable membrane 360. This invention attains this goal in thatthe membrane 360 is configured not horizontally, but at a slant to thehorizontal. The space 410 above the membrane can accordingly be filledwith electrophoretic buffer and thereupon an electrode, for instance 20b, that is acting as the anode, can be dipped into said space. Aftervoltage has been applied to the power leads 260, an electrophoreticexchange takes place between the volume of the reaction vessel and thevolume 410 above the membrane. As a result biological substance to beanalyzed is enriched between the gel 420 and the membrane 360 andsubsequently can be removed by means of a suitable pipet 290.

FIG. 9 shows apparatus such as in FIG. 7, however with a conductingreaction vessel and receptacle and also with a pipet tip with integratedelectrode.

FIG. 9 shows an especially simple apparatus of the invention wherein anelectrically conducting reaction vessel 270 illustratively serves as thecathode. An element 280 in the form of an electrically non-conductingcylinder part with a Y-branch can be moved by the appropriate transferelement 400 into the reaction vessel. The semi-permeable membrane 360 inthis instance is a non-horizontally integrated membrane 360 in such away that air bubbles are avoided underneath it. The withdrawal space 380can be loaded with the pipet tip in the manner previously discussed, andso can the space above the electrode 410. A gel with an overhang 420 ispresent in the transfer duct 150, whereby dipping it into the reactionliquid shall not create air bubbles. In this case an electricallyconducting pipet tip is simultaneously the electrode which then isdipped into the space above the semi-permeable membrane 410. Thereceptacle of the pipet tip 290 is made of an electrically conductingmaterial and in this manner can act as the electrical feed conductor tothe electrode. A corresponding receptacle for the reaction vessel 440may be provided in order to electrically feed the matching electrode.Using this apparatus, the following procedure can be carried out insimple manner using a corresponding xyz robot:

Initial condition gel element 280 in rack magazine step 1 transfer gelelement from rack in tube using the trans- fer device 400 step 2 fetchconducting pipet tip 290 step 3 pipet electrophoretic buffer ontomembrane 360 step 4 pipet electrophoretic buffer under membrane 360through aperture 380 step 5 dip pipet tip acting as electrode throughmembrane into space 410 step 6 guide electrophoresis by applying voltageacross elec- trodes 20a and 20b step 7 pipet eluate out of space 380

FIG. 14 shows a design of the invention of a conducting pipet tipwherein the electron current is improved by widening 450 the outer wallof the pipet tip. At the same this widening also may be used foragitation or mixing.

The following Examples elucidate the implementing modes of the method ofthe invention.

EXAMPLE 1

Measuring the Conductivity of the Electrodes of the Invention

The electrical conductivity was illustratively measured using conductingpipet tips (Canberra Packard, Dreieich, # 600 0604) or injection-moldedblanks of PRE-ELEC TP 4474 material (Premix Oy, Finland) using a digitalmultimeter (DT-890, Voelkner Elektronik, Brunswick, # 063-823-314).

Test Results resistance after about 1 min Initial resistance testingtime conducting pipet tips about 18 MΩ about 50 kΩ blanks 20 MΩ about 5MΩ

EXAMPLE 2

Coating an Electrically Conducting Plastic

2a Preparing Biotinylated Bovine Immimoglobulin G (R-IgG)

0.5 ml of a R-IgG solution [2 mg R-IgG (Boeluinger Mannheim Cat. No.1293621103 in 1 ml PBS (NaH₂PO₄*1H₂O 2.76 g/ltr; Na₂HPO₄*2h₂O 3.56g/ltr; NaCl 8 g/ltr; pH 7.25)] are mixed with 6 μltrD-biotinoyl-amincpronic acid- N-hydroxysuccinlmide ester solution in PBSand DMSO (batch per Biotin Labeling Kit, Boehringer Mannheim Order Nr.1418165) and agitated for 2.5 h at room temperature on a magneticstirrer and then are left to stand overnight. The molar ratio of biotinto R-IgG in this batch is 20/1.

2b Coating Electrically Conducting Plastic with Biotinylated R-IgG

A blank prepared by injection molding from PRE-ELEC TP 4474 (Premix Oy,Finland), is cut into disks of 4 mm diameter which are placed in a wellof an uncoated microtitration plate and are washed three times in asolution of 0.2 ml coating buffer (NaHCO₃, 4.2 g/ltr pH 9.6). Coating iscarried out over night by adding 0.2 ml coating buffer and 6 μμ/ltrR-IgG solution (2a).

Next the disks are washed 3 times with 0.25 ml of Milli-Q water eachtime and then are tested.

As a control, a batch of R-IgG lacking the biotinylation of 2a) isprocessed under identical concentrations.

2c Binding Tests

To test binding, the disks of 2b) are mixed with 200 μl of astreptavidin-peroxidase conjugate solution (Boehringer Mannheim CatalogNr. 1089 153; 1/20,000 dilution in PBS) and are incubated for 45 minwith agitation. Then the disks are separated by means of a magnetseparator (Boehringer Mannheim order # 1,641,794) and the residue isdiscarded. This procedure is repeated. Thereupon 200 μl of ABTS solution(Boehringer Mannheim Cat. Nr. 1 204 530 and 1 112 422) are incubated 15min. The disks are removed, the solution is transferred into amicrotitration plate (Innova GmbH) and measured at 405 nm.

Test results Disks with R-IgG biotin 453 mE Control with R-IgG 283 mE

EXAMPLE 3

Measurement of Electrochemiluminescence

Various materials were used for the electrodes 20 a, b in an apparatusshown in FIG. 11 where the plastic molded body 10 is a transparentpolystyrene tube (37 mm long, 12 mm in diameter) and the spacer 30 is apolypropylene tube (for instance a filter tube from the “High Pure PCRTemplate Preparation Kit” by Boehringer Mannheim order # 1 796 828, fromwhich the nonwoven was removed. On one hand electrodes made of aplatinum/ruthenium alloy wire of 0.5 mm diameter were used, and on theother hand electrodes approximately 3 mm×50 mm in size that were cut byknife from conducting pipet tips (Packard, Order # 6000604).

1 ml of a buffer containing tripropylamine such as the BoehringerMannheim Pro Cell buffer for the Elecsy product line (Order # 166 2988)were mixed with 5 μltr of a saturated solution oftris-(2,2′-bipyridyl)ruthenium(II) chloride hexahydrate (Sigma-Aldrich #93397 Fluka) and converted into (1). The apparatus was placed into thesample chamber of an LKB 1250 Luminometer, two electric feed wires beingpulled through extant apertures into the sample chamber. The apparatuswas further modified in that the last amplifier stage (IC 7) wasbypassed, resulting in smaller test signals. The test signals weredetected by a Digital Multimeter (DT 890, Voelkner Elektronik, Brunswick# 063 823 314). The electrode voltage source was an electrophoresistransformer (Hoelzel, Dorfen, # 0 628/1985). The luminometer wascalibrated using the internal standard and following the manual. Themixture of reagents at the platinum electrode was observed foaming verystrongly.

TEST RESULTS Test voltage at Relative light internal Trigger voltage Vintensity (mv) standard Platinum electrodes about 10 v max. 1,200 1Electrodes made of about 10 v max. 7 6 conducting plastic

In a variation, the voltage source for the plastic electrodes was theelectrophoresis transformer cited above (Hoelzel, Dorfen, #0 628/1985)and the voltage source for the platinum electrodes was a power pack madeby Voltcraft (NG-500, order # B518034) with a voltage divider and avoltage dropping resistor of 480 Ω to prevent foaming. The luminometerwas calibrated using the internal standard and following the manual, theparticular test values are each indicated.

TEST RESULTS test voltage at Trigger Trigger Relative light internalvoltage (v) current (ma) intensity (mv) standard (mv) Pt electrodes 3.251.1 max. 5,000 1 Conducting 25 0.175 max. 25 6 plastic electrodes 6.50.007 max. 7 6

In a third detection variation, the luminometer was modified in that thelast amplifier stage (IC 7) was bypassed, entailing test signals smallerby about a factor of 10. The test signals were directly entered in acomputer by means of a μM 4 measurement module (analog converter BMCSysteme GmbH, from Conrad Elektronic order # 10 75 57-99), the triggervoltage and the light emission in the form of the huninometer outputalso being stored simultaneously. The voltage source for the plasticelectrodes was an electrophoresis transformer (Hoelzel, Dorfen, #0628/1985) and for the platinum electrodes it was a power pack (NG 500Voltcraft, order # B5 18034) with a voltage divider and a voltagedropping resistor of 480 Ω to prevent foaming. The luminometer wascalibrated using the internal standard and following the manual. Whenusing the electrophoresis transformer and the analogue converter voltagedivider input voltage of 10 v was generated.

FIG. 21a is a plot of the test signal of the luminometer and of thetrigger voltage. FIG. 21b shows the dependency of the test signal on thetrigger voltage.

EXAMPLE 4a

Implementing Full Analysis of Nucleic Acids with Sample Preparation,Amplification and Measurement of Electrochemiluminescence Using GlassNonwoven Technology by Illustrating the Detection of the Hepatitis CVirus

This operation is based on apparatus shown in FIG. 19 though with onlyone permanent magnet 60 b in the immediate vicinity of thephotomultiplier 50, and on the control function shown in FIG. 2. A glassnonwoven is situated above the aperture 140 and was obtained from theQIAampBlood kit (cat # 29104) of Qiagen (Hilden). All associatedreagents from this kit used for sample preparation were used as follows:

200 μltr plasma were lysed following the accompanying instructions andbound to the glass nonwoven, all centrifugation operations beingreplaced by aspirating with a membrane pump 4151 (made by Eppendorf)through the aperture 140. 300 μltr of an electrophoretic buffer preparedin the manner of A T Andrews Electrophoresis, Clarendon Press, Oxford,1986, p 160) were used. The electrodes 20 a, b were wires of aplatinum-ruthenium alloy of 3 mm diameter and the voltage source was theelectrophoresis voltage source made by Hoelzel, Dorfen.

All the reagents of the HCV Amplicore Kit made by Hoffinann LaRoche(#075 3912,075 3890, 075 3904) were used for amplification. This kitemploys a biotinylated primer. The batch described by KKY Young et al inJournal of Microbiology 1993, pp 882-886 was appropriately adapted.

Thereupon 50 μltr of a corresponding amplification mix comprisingHCV-specific primers was added by pipetting through the aperture 130, acorresponding 7-fold concentration by the residual volume of about 300μltr for the individual concentrations were taken into account.Thereupon the temperature cycling routine described in the operationsfor use for RT-PCR was applied.

Next an HCV specific probe (nt 251-275) made in the manner of KKY Younget al (ibid) able to hybridize with the amplificate was added bypipetting through the aperture 140, the probe being labeled withruthenium. The temperature is raised to 45° C. and cooling to roomtemperature is implemented to melt the double strand and thehybridization with the probes. Thereupon 50 μltr streptavidin coatedmagnetic particles are added (Boehringer Mannheim order # 1 641 778) andare incubated for 15 min at 37° C. Next the permanent magnet 60 b wasactivated and all the solution of reaction was aspirated through theaperture 140. Thereafter 300 μltr of a buffer containing tripropylamine(Boehringer Mannheim Pro Cell buffer for the Elecsys product line order# 166 2988) was added by pipetting through the aperture 130 and atrigger voltage of 3.5 v was applied to the electrodes 20 a or 20 b. Theemitted light was measured by a photomultiplier as in Example 3.

EXAMPLE 4B

Carrying Out a Complete Nucleic-acid Analysis with Sample Preparation,Amplification and Electrochemiluminescence Measurement Using GlassParticles for an Example Detecting the Hepatitis C Virus

This example uses the apparatus of FIG. 19 with two permanent magnets 60a, b and the control function of FIG. 23.

All reagents are taken from the High Pure PCR template Preparation Kitof Boehringer Mannheim (order# 1 796 828). The glass magnetic particlesare made by Merck, Darmstadt (order # 1.01193.0001). 200 μltr plasmawere lysed according to accompanying instructions and bound to 50 μltrsuspension of glass magnetic particles. Next the magnet 60 a wasactivated and the lysing mixture was removed by evacuation using amembrane pump (made by Eppendorf, 4151) through the aperture 140. Theensuing washing steps were carried out similarly.

The ensuing electroelution and all other steps of further processingincluding detection were carried out as in Example 4a.

What is claimed is:
 1. An apparatus to isolate and enrich charged molecules, comprising: a plastic vessel defining a receiving chamber that receives a reaction mixture having at least one of a sample containing the charged molecules and reagents, said receiving chamber having an upper access aperture being externally accessible; a tube unit made of an electrically non-conducting plastic positioned within said receiving chamber, said tube unit having an open upper end and an open lower end opposite said open upper end, an inner surface of said tube unit contacting said reaction mixture in said receiving chamber wherein said tube unit is positioned inside the receiving chamber of said vessel through said upper access aperture, a withdrawal chamber and an electrode chamber located within said open upper end of said tube unit, said withdrawal chamber and said electrode chamber being separate from and adjacent to each other, said open upper end of said tube unit communicating with said open lower end of said tube unit by an internal chamber; a first electrode positioned within the receiving chamber and contactable with the reaction mixture inside the receiving chamber and outside the tube unit, and a second electrode positioned within said electrode chamber; and a semi-permeable membrane located at a lower end of said electrode chamber that prevents direct contact between the charged molecules and said second electrode.
 2. The apparatus as claimed in claim 1, wherein the tube unit is deposited on a base of the vessel.
 3. The apparatus as claimed in claim 1, wherein the tube unit comprises positioning arms that stabilize a position of the tube unit in the vessel.
 4. The apparatus as claimed in claim 1, wherein a gel material is provided in the internal chamber of the tube unit and extends completely across an interior width of said tube unit.
 5. The apparatus as claimed in claim 1, wherein the tube unit flares away from the second electrode in a direction toward a base of the vessel.
 6. The apparatus as claimed in claim 1, wherein at least one of the first and second electrodes is made of a plastic comprising electrically conducting additives.
 7. The apparatus as claimed in claim 1, wherein at least one of the first and second electrodes is a separate element from the vessel.
 8. The apparatus as claimed in claim 1, wherein at least one of the first and second electrodes is designed as either one of a pipet tip and a part of a pipet tip.
 9. The apparatus as claimed in claim 1, further comprisina attachments that are either mounted on an exterior of the second electrode or integrated with the exterior of the second electrode.
 10. The apparatus as claimed in claim 1, wherein at least one of the first and second electrodes is integral with a wall of the vessel.
 11. The apparatus as claimed claim 1, wherein at least one of the first electrode, the second electrode, and the semi-permeable membrane is provided with a coating.
 12. The apparatus as claimed in claim 11, wherein the coating comprises several layers.
 13. The apparatus as claimed in claim 11, wherein the coating includes at least one biological polymer.
 14. The apparatus as claimed in claim 13, wherein the biological polymer is able to bind a nucleic acid.
 15. The apparatus as claimed in claim 1, wherein adsorbents are either present in the vessel or directed into the vessel to separate the charged molecules.
 16. The apparatus as claimed in claim 15, wherein the adsorbents include at least one of a silica gel, and an agarose gel, and a polyacrylic amide gel, and an ion-exchanger substance, and a fiberglass nonwovens, and glass particles, and glass enclosed magnetic particles.
 17. The apparatus as claimed in claim 1, wherein a magnetic field source associated with the vessel and can be transferred between an active state wherein the magnetic field source exerts an attractive force on magnetic particles present in the vessel and an inactive state wherein magnetic field source exerts substantially little or no force on the magnetic particles.
 18. The apparatus as claimed in claim 17, wherein the magnetic field source comprises an electromagnet.
 19. The apparatus as claimed in claim 1, further comprising a pipetting robot.
 20. The apparatus as claimed in claim 17, wherein a pipetting robot and the magnetic field source are integrated so that the pipetting robot is able to manipulate the magnetic field source between the active and inactive states.
 21. The apparatus as claimed in claim 19, wherein the pipetting robot includes a gripper.
 22. A combination structure of the apparatus as claimed in claim 1, wherein the second electrodes of the apparatus are interconnected, the first electrodes of the apparatus are interconnected, and a plurality of the vessels are combined into a microtitration plate.
 23. A method to isolate and/or analyze charged molecules, by adsorbing charged molecules in solution to an appropriate adsorbent, by separating the remaining solution and optionally washing the adsorbent, detaching the molecules from the adsorbent and separating the molecules, wherein the method is carried out in an apparatus as claimed in claim 1, the apparatus being used to contain the adsorbent, or an absorbent previously loaded with charged molecules being introduced into the apparatus and wherein detachment of the molecules from the adsorbent is implemented by electroelution.
 24. A method to isolate and/or analyze charged molecules by depositing a sample comprising the charged molecules on a separation gel and by electrophoretically separating the charged molecules based on different molecular weights, wherein the method is carried out in an apparatus as claimed in claim 1, the apparatus comprising a separation gel introduced into the apparatus before the sample is deposited.
 25. A method to isolate and/or analyze charged molecules by adsorbing the charged molecules in solution to an appropriate adsorbent, by separating the remaining solution and optionally washing the adsorbent, detaching the molecules from the adsorbent and separating the molecules, wherein the method is carried out in a combination structure as claimed in claim 22, the combination structure being used to contain the adsorbent, or an absorbent already loaded with charged molecules being introduced into the combination structure, and wherein at least the detachment of the charged molecules from the adsorbent is implemented by electroelution.
 26. A method to isolate and/or analyze charged molecules by depositing a sample comprising the charged molecules on a separation gel, and by electrophoreuically separating the charged molecules based on different molecular weights, wherein the method is carried out in a combination structure as claimed in claim 22, the combination structure comprising a separation gel introduced into the combination structure before the sample is deposited. 